Hill Reaction of Isolated Chloroplasts Flashcards
What is the Hill reaction
A light-driven reaction where isolated chloroplasts reduce added oxidants and evolve oxygen by splitting water
What is the balanced equation for the Hill reaction
H₂O → ½ O₂ + 2H⁺ + 2e⁻
In vivo, what happens to the electrons from water splitting
They are transferred down the electron transport chain and reduce NADP⁺ to form NADPH
What is DCPIP
A blue artificial electron acceptor that becomes colourless when reduced
What reduces DCPIP in the Hill reaction
Electrons from plastoquinone (PQ) in the electron transport chain
What are detergents
Amphiphilic molecules with both hydrophobic and hydrophilic regions used to disrupt membranes, solubilise lipids/proteins, and denature proteins
What is SDS and what does it do
SDS is a strong anionic detergent that denatures proteins by binding their hydrophobic core and pulling them into solvent via its charged sulphate group
What is Triton-X-100 and how is it different from SDS
A milder, non-ionic detergent that solubilises lipids while preserving protein structure and interactions
Why would you choose Triton-X-100 over SDS
To extract membrane proteins in their functional form without denaturing them
Is protein denaturation by heat always permanent
No, it can be reversible or irreversible depending on the temperature and exposure duration
What are chemical inhibitors
Small molecules that bind specific biological targets to inhibit their function, used in research and pharmacology
How do chemical inhibitors help study biological pathways
By selectively blocking steps in a pathway, allowing identification of the order and function of pathway components
What are the two conditions needed to study a pathway with inhibitors
- A measurable intermediate in the pathway.
- A specific inhibitor targeting a known step.
Why are multiple inhibitors useful
They allow detailed dissection of pathways by pinpointing where each target lies relative to specific intermediates
What does the Hill reaction demonstrate about chloroplasts
That they contain the necessary components to carry out light-dependent electron transfer reactions independently
Why is oxygen evolution a good indicator of electron transport activity
Because splitting water (photolysis) releases oxygen, electrons, and protons—so O₂ evolution directly reflects electron flow
What would happen if light was excluded from a Hill reaction setup
No electron transfer would occur, and DCPIP would remain blue (oxidised), because chloroplasts require light to drive photolysis.
How do detergents help study membrane proteins
They isolate proteins from membranes while maintaining their structure (in mild detergents) or denature them for structural studies (in strong detergents like SDS)
What’s the purpose of SDS in SDS-PAGE
SDS denatures proteins and imparts a uniform negative charge, allowing them to be separated by size during electrophoresis
What are some signs of protein denaturation in a biological experiment
Loss of function, loss of solubility, aggregation, or failure to catalyse a reaction
What makes a good chemical inhibitor for pathway analysis
High specificity, known mechanism of action, and reversibility (if needed for recovery experiments)
If a detergent-treated chloroplast still reduces DCPIP, what does this imply
The detergent did not fully disrupt the electron transport chain or denature the proteins involved
Why is light energy required for the Hill reaction
To excite electrons in photosystem II, enabling the splitting of water and transfer of electrons down the chain
Why is it important to keep chloroplasts on ice before an experiment
To preserve enzyme and membrane function by preventing denaturation or degradation
